The objective of this work is to understand the physiological role of enzymatic protein carboxyl methylation reactions in human tissues and other cells. We propose to continue our studies of an L-isoaspartyl/D-aspartyl methyltransferase (E.C. 2.1.1.77) that catalyzes the modification of damaged proteins containing these unusual residues. This enzyme is found in the cytosolic fraction of all mammalian tissues examined and recognizes the altered residues that result from spontaneous racemization, isomerization, and deamidation of normal L-aspartyl and L- asparaginyl residues in aged proteins. In model systems, the formation of methyl esters at L-isoaspartyl residues can lead to their conversion to L-aspartyl residues and suggests that this enzyme functions to repair certain types of covalent damage to intracellular proteins and limit their accumulation in aging cells. We will use a combination of biochemical and molecular biological techniques to characterize the human gene for this enzyme and its polymorphisms, to delineate the three- dimensional structure of the human enzyme, and to analyze residues responsible for binding and methylation of damaged protein substrates. We are also interested in examining the effects of enzyme overproduction and underproduction in transgenic mice. Since the structure of this enzyme has been remarkably well conserved from bacteria to human cells, we will also utilize genetically-manipulatable model systems, including bacteria and the nematode warm Caenorhabditis elegans. This methyltransferase may represent one of the first members of a class of enzymes that can check spontaneous damage to cellular proteins, and its disruption in pathological conditions may both decrease their useful lifetime and contribute to accelerated aging processes. Our recent discovery of a new class of protein carboxyl methyltransferase suggests that these enzymes may also have roles in the regulation of cellular metabolism including cell cycle control in tumor formation. In 1993, we found that one of the major cellular protein phosphatases (2A), which plays an essential role in modulating enzyme activity by reversing the action of various protein kinases, is a specific substrate for a novel C-terminal leucine methyltransferase. In this grant period, we will continue our investigation of this C-terminal leucine methylation reaction. We will purify and characterize the enzymes involved in these reactions from both mammalian tissues and yeast, and characterize potential demethylases as well. The ability to pharmacologically control these modification enzymes may represent new therapies for cancer and other diseases. Finally, we are interested in searching for additional new types of protein carboxyl methyltransferases in cells that may play unique roles in metabolism or signaling reactions.